Life Cycle, Pathogenicity and Prophylaxis of Plasmodium vivax

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1 Discipline Courses-I Semester-I Paper: Divesity and Evolution of Choradata-I Lesson: Life Cycle, Pathogenicity and Prophylaxis of Plasmodium vivax Lesson Developer: Anubha Das College/Department: Zakir Husain College, University of Delhi Institute of Life Long Learning, University of Delhi 0

2 Table of Contents Introduction Discovery Taxonomic position Geographical distribution Host Reservoir Habit and habitat Life cycle of P. vivax The Human phase Exoerthrocytic stage Erythrocytic stage The mosquito phase: Sporogony Pathogenecity Incubation period Initial symptoms Malarial paroxysm Relapse Epidemiology Mode of infection Infective stage Source of infection Transmission ways Factors affecting transmission Susceptible population Prophylaxis Treatment Summary Exercises Glossary References Introduction Institute of Life Long Learning, University of Delhi 1

3 Malaria parasites belongs to genus Plasmodium, are classified in the domain Eukarya, kingdom Protista, Phylum Apicomplexa. All members of the phylum Apicomplexa are obligate intracellular parasites of vertebrates (humans, birds, lizards, rodents, and primates) causing considerable damage and even leading to death of the host. Fig. 1. Cycle of Malaria Source: ILLL in house Life cycle of a typical sporozoan (class Sporozoea) includes 3 distinct stages: sporogony, schizogony (or merogony) and gamogony (or gametogony) and there is alternation between an invertebrate (mosquito) and a vertebrate (lizard, bird, mammal) host. The disease is widespread in tropical and subtropical regions in a broad band around the equator, including much of Sub-Saharan Africa, Asia, and the Americas. Plasmodium is an ancient organism and it is been with us since long. Value addition: Ronald Ross s poem! Heading text: The heart of poet and brain of a scientist! Body text: Ronald Ross penned an optimistic poem on August 21, 1897, shortly afte discovering malaria parasites in mosquitoes: The poem unfortunately overstated the prospect for a victory over malaria as, we after 113 years, are still fighting to control the deadlies disease. This day relenting God Institute of Life Long Learning, University of Delhi 2

4 Hath placed within my hand A wondrous thing; and God Be praised. At His command, Seeking His secret deeds With tears and toiling breath, I find thy cunning seeds, O million-murdering Death. I know this little thing A myriad man will save. O Death, where is thy sting? Thy victory, O Grave! Source: Value addition: Video Heading text: No ordinary mosquito bite! Body text: No ordinary mosquito bite! Source: Discovery The genus Plasmodium was described in 1885 by Ettore Marchiafava and Angelo Celli. At present, more than 200 species of this genus are identified. It is also known as the malarial parasite. Out of the over 200 known species of Plasmodium, at least 11 species infect humans. Other species infect other animals, including monkeys, rodents, birds, and reptiles. The parasite always has two hosts in its life cycle: a vector usually a mosquito and a vertebrate host. Plasmodium falciparum (Welch, 1897), Plasmodium vivax (Grassi e Feletti, 1890), Plasmodium malariae (Laveran, 1881) and Plasmodium ovale (Stephens, 1922) are the four species of Plasmodium known to cause malaria in human. The deadliest of the four species of the parasite is Plasmodium falciparum. Value addition: Did you know? Heading text: A fifth species of Plasmodium hosted by man! Body text: A new species of Plasmodium has been discovered in Malaysian Borne Plasmodium knowlesi, which jumps the species barrier, spreading from monkeys to humans Institute of Life Long Learning, University of Delhi 3

5 So there are now five species of malaria parasites that can be hosted by humans. Source: Institute of Life Long Learning, University of Delhi 4

6 Value addition: (Biographic sketch) An inspiring story.hard work and consistent approach really pays off! Heading text: Sir Ronald Ross and the Discovery that Mosquitoes Transmit Malarial parasite. Body text: Sir Ronald Ross was born to Sir C.C.G. Ross, a General in the Indian Army and his wife Matilda in 1857 in Almora, India. He was sent to England for education at the age of eight where he spent much of his childhood with an aunt and uncle. During his early years he developed interests in poetry, literature, music, and mathematics, all of which he continued to engage in for the rest of his life. In 1881, he returned to India, as medically qualified (MRCS and LSA) and joined the Madras branch of the Indian Medical Service. In 1892, Ross became interested in malaria even though originally he doubted the parasite s existence. In 1894, Patrick Manson, considered by many to be the father of tropical medicine, showed him 'Laveran's bodies' (malaria parasites), and convinced him of the possibility that mosquitoes carried malaria. Eventually he became an enthusiastic convert to the belief that malaria parasites were in the blood stream. On returning to India, Ross rejoined his regiments at Secunderabad, where he started breeding mosquitoes and fed them on malaria patients in order to experimentally prove his theory. At the end of May 1896, on the basis of his observation he wrote to Manson: "The belief is growing on me that the disease is communicated by the bite of the mosquito... She always injects a small quantity of fluid with her bite - what if the parasites get into the system in this manner." Unfortunately, he could not prove his theory as he was using Culex mosquitoes, which do not transmit malaria. After his return to Secunderabad, Ross decided to continue his investigations with mosquito species from a highly malarious area. He went to the notoriously malarious valley of Sigur Ghat and three days later he contracted the disease, despite having slept under a mosquito net and behind closed windows. After returning to the Ghat, he got interested in mosquito species he had not seen before. In mid-august 1897, unfamiliar larvae brought by his assistants hatched into similar mosquitoes that Ross had found in Sigur Ghat. Ross could not find anything after dissecting the mosquito. They were fed on a patient called Husein Khan but nothing was found when they were dissected. More mosquitoes hatched out and were fed, and by 20 August 1897 Ross was down to his last two mosquitoes. He dissected one and found nothing - until he got to the stomach, "when I saw a clear and almost perfectly circular outline before me of about 12 microns in diameter. The outline was much too sharp, the cell too small to be an ordinary stomach-cell of a mosquito. I looked a little further. Here was another, and another exactly similar cell." The following day Ross killed his last mosquito and found similar but much larger cells. He Institute of Life Long Learning, University of Delhi 5

7 wrote to Manson with his exciting news: "Now prick up your ears because the hunt is up again." Ross got his work verified by a colleague, Surgeon-Major John Smyth and took ten days' leave to write a paper, 'On some peculiar pigmented cells found in two mosquitoes fed on malarial blood'. He sent this off immediately to the British Medical Journal, which took three months to publish it. By that time, Ross had been transferred to a small and isolated station called Kherwara in Rajastan, which was conspicuously free of malaria. But he continued his research into malaria in India, using birds as experimental model. By July 1898, he had demonstrated that mosquitoes could serve as intermediate hosts for bird malaria. He found that the malaria parasites in the mosquitoes migrate to the insects' salivary glands, which gets transmitted to other birds by bite of infected mosquito. He sent a copy of his report 'On the cultivation of Proteosoma labbe 1898' written for the Director General of the Indian Medical Service to Patrick Manson. In the Annual Meeting of the British Medical Association in Edinburgh, Manson presented report on Ross's work on the oocysts in mosquitoes' stomach and also his telegraph, which stated that the parasite was transferred through the vector's bite. In 1902 he became the first Briton to win the Nobel Prize in Physiology and Medicine. He died, after a long illness, at the Ross Institute on 16 September Source: and Value addition: Interesting to know! Heading text: An ordinary looking building with an extra-ordinary past! Body text: Institute of Life Long Learning, University of Delhi 6

8 Source: and Taxonomic position Kingdom: Protista Phylum: Apicomplexa Class: Sporozoa Genus: Plasmodium NOTE: Vth ed. Barnes book has been consulted for classification Geographical Distribution Table 1. Geographical distribution of four species of Plasmodium P. vivax P. falciparum P. malariae P. ovale Widespread in tropical and subtropical areas of Central and South America, India and South East Asia. Range extends into temperate areas Relatively uncommon in Africa Widespread in tropical and subtropical areas of Central and South America, Africa and South East Asia Tropical and subtropical areas of Central and South America, Africa and South East Asia Primarily tropical Africa, especially Sub Saharan Africa Occurrence of malaria depends mainly on climatic factors such as temperature, humidity, and rainfall. Malaria is more prevalent in tropical and subtropical areas, where Anopheles mosquitoes get optimum condition for survival and propagation. Malaria parasites can complete their growth cycle in the mosquitoes ("extrinsic incubation period"). Temperature is particularly critical in case of malaria. In many malaria-endemic countries, malaria transmission does not occur in all parts of the country and also may not occur throughout the year. Transmission will not occur At very high altitudes During colder seasons in some areas In deserts (excluding the oases) Institute of Life Long Learning, University of Delhi 7

9 That is why in areas closer to the equator Transmission is more intense Malaria is transmitted year-round. The highest transmission is found in Africa South of the Sahara and in parts of Oceania such as Papua New Guinea. In cooler regions, transmission will be less intense and more seasonal. There, P. vivax might be more prevalent because it is more tolerant of lower ambient temperatures. In many temperate areas, such as Western Europe and the United States, malaria has been eradicated through economic development and public health measures. However, most of these areas have Anopheles mosquitoes that can transmit malaria, and reintroduction of the disease is a constant risk. Host Fig.2. Malaria-endemic countries Source: The vertebrate host include human, monkeys, rodents, birds, and reptiles. Vector Institute of Life Long Learning, University of Delhi 8

10 In the life cycle of Plasmodium, a female Anopheles mosquito (the definitive host) transmits a motile infective form (called the sporozoite) to a vertebrate host such as a human (the secondary host), thus acting as a transmission vector. All the Plasmodium species causing malaria in humans are transmitted by mosquito species of the genus Anopheles. There are seven species of Anopheles mosquito found to cause malaria in humans in India. These are: A. culicifacies A. stephensi A. phillippinensis A. sundaicus A. fulviatilis A. maculatus A. minimus Both sexes of mosquitoes live on nectar. Because nectar's protein content alone is insufficient for oogenesis (egg production) one or more blood meals is needed by the female so only female mosquitoes bites. Value addition: Interesting fact Heading text: Malarial vector Body text: Species of the mosquito genera Aedes, Culex, Culiseta, Mansonia an Theobaldia can also transmit malaria but not to humans. Bird malaria is commonl carried by species belonging to the genus Culex. Ross who worked with species from the genus Culex discovered the life cycle of Plasmodium. Source: ILLL in house Reservoir Symptomatic as well as tolerant humans with high parasitemia count to infect mosquitoes. A person with gametocytes in the circulation is a carrier or reservoir. Gametocytes do not develop further or divide in the vertebrate host and unless taken up by the vector mosquito, they die in a few days. Life cycle and Morphology of Plasmodium vivax Life cycle of Plasmodium is divided into two phases: Asexual phase: occurs in humans (intermediate host of Plasmodium). Sexual phase: occurs primarily in the female Anopheles mosquito (definitive host of Plasmodium). This alteration of generation i.e. alteration of asexual phase and sexual phase is in complete synchronization with alteration of hosts. The asexual phase taking place in the human is followed by the sexual phase in the mosquito. As the sexual phase is completed in the mosquito, it is considered as the definitive host of Plasmodium parasite. Human beings are the intermediate host as the asexual multiplication takes place here. Institute of Life Long Learning, University of Delhi 9

11 Fig.3. Life cycle of Plasmodium The malaria parasite life cycle involves two hosts: Anopheles mosquito and Human where the parasite complete its sexual and asexual phase of lifecycle respectively. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host Sporozoites from the blood capillaries goes into hepatocytes and mature into hepatic schizonts through exoerythrocytic schizogony, which rupture and release merozoites. (In P. vivax and P. ovale a dormant stage [hypnozoites] can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later.) After this initial replication in the liver (exo-erythrocytic schizogony ), the hepatic merozoites released from liver cells invade RBC and undergo asexual multiplication (erythrocytic schizogony) Merozoites infect red blood cells schizonts, which rupture releasing merozoites. The ring stage trophozoites mature into erythrocytic. Some parasites differentiate into sexual erythrocytic forms (gametocytes). Blood stage parasites are responsible for the clinical manifestations of the disease. The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood meal parasites multiplication in the mosquito is known as the sporogonic cycle. The. While in the mosquito's stomach, the microgametes penetrate the macrogametes generating zygotes Institute of Life Long Learning, University of Delhi 10

12 The zygotes in turn become motile and elongated (ookinetes) of the mosquito where they develop into oocysts sporozoites which invade the midgut wall. The oocysts grow, rupture, and release, which make their way to the mosquito's salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle Source: Table 2. Various morphological form of Plasmodium vivax S.N o Stage Morphology Type Host Infected organ 1. Sporogony Sporozoite (Asexual form) 2. Exoerythrocytic schizogony or Merogony 3. Exoerythrocytic schizogony or Merogony Exoerythrocy tic schizont or meront (Asexual form) Merozoite (Asexual form) Infective Mosquito Salivary gland Noninfective Invades Inoculated in human during blood meal from where they invade hepatocyte Human Liver Non-invasive Infective Human Liver Invades erythrocyte Image 4. Exoerythrocytic schizogony or Merogony 5. Erythrocytic schizogony 6. Erythrocytic schizogony 7. Erythrocytic schizogony Hypnozoite (Asexual form) Signet ring stage (Asexual form) Trophozoite (Asexual form) Schizont (Asexual form) Dormant Human Liver Relapse infections of malaria Diagnostic Human RBC Non-invasive feeding stage Diagnostic Human RBC Non-invasive feeding stage Diagnostic Human RBC Non-invasive 8. Erythrocytic schizogony Gametocyte (Sexual form) Diagnostic Human RBC Non-invasive 9. Sporogony Ookinete (Asexual form) Diagnostic Mosquito Gut wall epithet-- lium Non-invasive Institute of Life Long Learning, University of Delhi 11

13 Source: ILLL in-house The Human Phase Blood meal acts as a protein source for egg production in female mosquitoes. In the female mosquitoes, there is a continuous cycle of blood meal and oviposition. The female mosquito repeatedly contacts the vertebrate host, ingests the malarial parasites, followed by the multiplication and maturation of the parasites inside the mosquito, and their transmission to other individual hosts during subsequent feedings. Human infection comes through the bite of the infective female Anopheles mosquito. The sporozoites, which are infective forms of the parasite, are present in the salivary glands of the mosquito. They are injected into blood capillaries when the mosquito feeds on blood after piercing the skin. Value addition: Did you know? Heading text: Plasmodium and Nobel prize! Body text: Charles Alphonse Laveran, a French military doctor, won Nobel Prize i 1907 for the discovery of the malarial parasite, Plasmodium, in human blood sample (1880). He donated the prize money to the Pasteur Institute for the creation of laboratory devoted to tropical diseases. He died in Paris on May 18, Source: Fig.4. Anopheles stephensi mosquito is obtaining a blood meal from a human host through its pointed proboscis. Note the droplet of blood being expelled from the abdomen after having engorged itself on its host s blood. This mosquito is a known malarial vector with a distribution that ranges from Egypt all the way to China. Source: Institute of Life Long Learning, University of Delhi 12

14 Fig.5. A female mosquito inoculating the human host with sporozoites Around sporozoites are injected at a time but even hundreds may be introduced in case of heavy infections. The sporozoites pass into blood stream, where many are destroyed by the phagocytes, but some reach the liver and enter the hepatocytes (liver cells). Exo-erythrocytic (Tissue) Stage Fig.6. Process of Exo-erythrocytic Schizogony. Source: ILLL in house Institute of Life Long Learning, University of Delhi 13

15 Fig.7. Sporozoites making an entry and developing in a hepatocyte Source: Here, it undergoes a process of asexual replication to give rise to a schizont. During this preerythrocytic schizogony or merogony the sporozoites that are elongated spindle-shaped bodies change the shape and tend to become somewhat rounded inside the liver cells. The parasite invades and develops in a nucleated liver cells of host. The sporozoites grow in size and undergo repeated nuclear division to form several daughter nuclei, each of which is surrounded by cytoplasm. This stage of the parasite is called the pre-erythrocytic or exoerythrocytic schizont or meront. The hepatocyte is distended by the enlarging schizont and the liver cell nucleus is pushed to the periphery and there are no pigments in liver schizonts, unlike erythrocytic schizogony. Fig.8. A developing schizont in hepatic cell Source: At the end of the pre-erythrocytic stage of 6-16 days (depending on the Plasmodium sp., after the time of infection), the cell containing the schizont ruptures releasing the merozoites into Institute of Life Long Learning, University of Delhi 14

16 the surrounding tissue and blood circulation. While some are phagocytized,most of them invade the red blood cells present in the sinusoids of the liver. Fig.9. The exit from liver cells Source: The pre-erythrocytic phase is asymptomatic phase. Neither any clinical symptoms nor any pathological damage is produced by the parasite in this pre-erythrocytic phase. Blood is sterile and the parasite is not susceptible to drugs in the tissue phase. The size of mature schizont and the number of merozoites produced also vary with the species of parasite. The merozoites enter the blood stream and infect erythrocytes by a process of invagination. The interval between the entry of the sporozoites into the body and the time when malaria parasites first appears in the blood is called pre-patent period. It is different from the incubation period, which is the first appearance of clinical symptoms after infection. In the human malarial species, it was originally thought that there are two successive preerythrocytic cycles, but it is now known that in two species of human malarial parasite i.e., Plasmodium vivax and P. ovale some sporozoites multiply inside hepatocytes promptly to form schizonts i.e., some of the liver sporozoites immediately start the exo-erythrocytic schizogonic cycle, while others remain in the liver as hypnozoites (Greek hypnos-sleep; zoon= animal). Hypnozoites can remain dormant in the liver for months to years developing into infective form leading to relapse infections. This is called secondary exo-erythrocytic cycle.the triggers that activate the hypnozoites to become schizonts and release merozoites are unknown. Erythrocytic Stage It was believed earlier that the merozoites released from a schizont directly penetrate the membrane of a red blood cell and then develop but now it has been established that the merozoites enter erythrocytes by endocytosis. This key event in the malarial life cycle has been extensively examined, as the inhibition of this process can be regarded as a crucial goal in the development of a malarial vaccine. The invasion of erythrocyte involves recognition and attachment of the merozoites to the erythrocyte membrane. If, on initial contact with an erythrocyte, the apical complex end of the Institute of Life Long Learning, University of Delhi 15

17 parasite is not directed towards the blood cell surface, a re-orientation takes place. After reorientation, some deformation of the erythrocyte membrane may occur and invagination and endocytosis follows. Once merozoites invade red blood cells, they feed on the haemoglobin and divide mitotically to form an erythrocytic schizont. The merozoites are pear-shaped bodies and enter the RBCs by endocytosis by creating a pit on RBCs membrane. The process of entry into the RBC takes about 30 seconds. Value addition: Interesting to know! Heading text: What is so special about the proteins present on Red Blood Cells? Body text: The receptor for merozoites is glycophorin, a major glycoprotein on the red cell. The differences in the glycophorins of red cells of different species may account for the species specificity of malaria parasites. Source: Textbook of Medical Parasitology, Paniker, C.K.J 6 TH ed After making its entry into RBC the merozoites rounds up and looses its internal organelles (especially apical complex). The feeding stage of parasite is called trophozoite (trophosgrowth). The parasite, inside RBC has a central vacuole and cytoplasm is pushed on the periphery with nucleus situated at one pole. When stained with Giemsa stain, the cytoplasm is stained blue and nucleus red while the central vacuole remains unstained giving the parasite an annular or signet ring appearance. These young parasites are therefore called the ring forms or early trophozoites. It grows at the expense of the erythrocyte. The parasite feeds on the haemoglobin of the erythrocyte but doesn t metabolise it completely leaving behind a haematin-globin pigment as residue called as the malarial pigment (earlier known as haemozoin pigment). These iron containing pigments keep on accumulating in the body of parasite as dark granules and becoming more prominent with time as the parasite grows. Fig.10. Signet ring forms Plasmodium vivax trophozoites inside a red blood cell Source: Institute of Life Long Learning, University of Delhi 16

18 Fig.11. Cycles of asexual parasite replication inside red blood cells. throcyte within seconds (1), initiating a new erythrocytic cycle. Source: ILLL in house Institute of Life Long Learning, University of Delhi 17

19 Value addition: Interesting to know! Heading text: The sickle cell trait provides advantage in malaria endemic regions Body text: In areas where malaria is endemic, sickle cell trait provides a survival advantage over people with normal hemoglobin. People particularly children infected with P. falciparum are more likely to survive the acute illness if they have a sickle cell trait. The reason for resistance seems to be that as plasmodium parasites grow in the erythrocytes, they lower the intracellular ph and generate hydrogen peroxide. The lower ph promotes sickling of the erythrocytes and the hydrogen peroxide damages cell membranes, which become more permeable to K +. the resulting intracellular decrease in K + kills the parasite. Source: Ahmed,N., Dawson, M., Smith, C and Wood, Ed., Biology of Disease. The malarial pigment is released when the parasitized cells rupture and is taken up by the reticuloendothelial cells. The ring form becomes irregular in shape as it develops further and enlarges in size. This is called the amoeboid form or late trophozoite. On the inner surface of the erythrocyte the membrane of developing parasite starts accumulating and gives the appearance of clefts on the erythrocyte surface. After a period of growth, the trophozoite divides asexually by process of erythrocytic schizogony. Division of nucleus 3-5 times produces a variable number of small nuclei. This stage of parasite is called the schizont or meront (formerly known as segmenter or rosette forms). Initially, the nucleus divides but the cytoplasm remains undivided, this stage is referred to as early schizont. This is followed by the division of cytoplasm, surrounding each daughter nucleus and forming a late schizont. The mature schizont is the fully-grown form, in which a number of small merozoites are seen, each having a nucleus with surrounding cytoplasm. After the completion of schizogony, the red cell bursts and the merozoites are released into the blood stream. Value addition: Activity Heading text: Identifying the various stages of malarial parasite. Body text: In the laboratory, try to locate and identify the various stages of malarial parasite present in a blood film from a person infected with Plasmodium sp. Source: self created (author) Institute of Life Long Learning, University of Delhi 18

20 Fig.12. Structure of a merozoite (showing various antigens) Source: The merozoites then invade fresh red cells to produce another generation of parasites. This repeated erythrocytic cycle of schizogony leads to a progressive increase of parasitaemia until immune system of the host body slows down this process. Value addition: Interesting facts Heading text: Merozoites show a distinct preference for erythrocytes of a certain age Body text: In some species, merozoites show a distinct preference for erythrocytes o a certain age. Many avian species attack almost exclusively the young erythrocytes. I the human plasmodia, the merozoites of P. vivax attack young immature corpuscle (reticulocytes) whereas those of P. malariae attack older ones, and those of P. falciparum indiscriminately enter any available. This partly explains the virulence of P. falciparum infections in which 10 percent or more of the erythrocytes may be attacked, whereas P vivax seldom occurs in even 1 percent of the corpuscles and P. malariae seldom in mor than 0.2 percent. Source: Author Institute of Life Long Learning, University of Delhi 19

21 All schizonts release their merozoites within a period of several hours together with a pigment and other waste products, and there is a sudden paroxysm of fever in the host, characterised by a marked rise in temperature. This periodicity is one of the most remarkable features of malarial organisms.the length of cycle is of 48 hours in P. vivax. After a few cycles of erythrocytic schizogony, some merozoites that infect red blood cells do not proceed to become schizonts, but instead develop into sexually differentiated forms, the gametocytes. The precise mechanism for this differentiation remains unknown but it is thought that the development of host antibodies may play a large part in the process, other factors include stress due to rising parasitaemia, the effects of drug suppression, and nutrient depletion. The gametocytes grow in size till they almost fill the red blood cell, but the nucleus remains undivided. Development of gametocytes generally takes place within the internal organs such as spleen and bone marrow, and only the mature forms appear in circulation. The mature gametocytes are round in shape and female gametocyte is always larger (macrogametocyte) than male gametocyte (microgametocyte). A person with gametocytes in the circulation is a carrier or reservoir. Gametocytes do not develop further or divide in the vertebrate host and unless taken up by the vector mosquito, they die in a few days. The process of differentiation of gametocytes is called gametocytogenesis. Value addition: Video Heading text: Plasmodium Life cycle in Human Body text: Life cycle of Plasmodium in Human host Source: The Mosquito Phase During blood meals taken from persons infected with malarial parasites, the mosquitoes pick up both the asexual and sexual forms of parasite. The asexual forms are digested, but the gametocytes are set free in the stomach and undergo further development. The cells rapidly undergo gamete production. This marks the beginning of gametogenesis and sexual cycle. A reorganization in the macrogametocyte results in the formation of the female gamete. The formation of male gametes is however an explosive phenomenon and the ultrastructural changes are rapid and stunning. A complex cytoplasmic reorganization takes place, involving rapid nuclear divisions, which result in the production of 8 motile haploid gametes each provided with an actively motile, whip-like filament (flagella). These flagella, which are male gametes (microgametes) lash about for sometime and then break free. This process of male gamete formation from gametocyte is called exflagellation. Value addition: common misconception Heading text: There is a difference between Gametocytogenesis and Gametogenesis Body text: It is not unusual to get confused between the two terms though there exists a huge difference between the two. The formation of gametocytes or cells which give rise to gametes is known as gametocytogenesis and in case of the Institute of Life Long Learning, University of Delhi 20

22 malarial parasite the process takes place inside the human body while the formation of gametes from gamete producing cells is known as gametogenesis and in case of malarial parasite this happens inside the body of mosquito host. Source: Fig.13. Ultrastructure of exflagellated Plasmodium microgametocytes Source: COPYRIGHT SANOFI ALL RIGHTS RESERVED. A change from a warm blooded animal to a cold blooded animal, ph and redox potential are the factors which are responsible for the gametogenesis to take place. Once a microgamete fertilises a macrogamete (which happens in half to two hours after the blood meal), the resulting zygote develops into a motile elongated ookinete and develops an apical complex, anteriorly. The ookinete penetrates the gut wall of the mosquito and develops as an oocyst between the epithelium and the basement membrane. It changes the shape and becomes round with an elastic membrane. This stage is called oocyst. Ronald Ross discovered these pigmented oocysts in the stomach of mosquito for the first time, a discovery that established the mosquito transmission of malaria. Institute of Life Long Learning, University of Delhi 21

23 Fig.14. Oocysts on the stomach wall of a mosquito Source: The oocyst matures in days depending upon temperature and species. The chromatin divides repeatedly. This sporogony leads to the development within the oocyst of about a thousand sporozoites, each with a central nucleus and an anterior apical complex. Fig.15. Sporozoites after dissection of an infected mosquito Source: COPYRIGHT SANOFI ALL RIGHTS RESERVED. The mature oocyst bulges into the body cavity of the mosquito, and when it ruptures the sporozoites enter the haemocoel. The sporozoites reach the salivary glands of the mosquito and enter the salivary duct. The time taken to complete sporogony in mosquito is about 1-4 weeks; depending on the temperature and the species. The mosquito is now infective and when it feeds on humans, the sporozoites are injected into the skin to initiate human infection and a new cycle begins. Period of time required for the development of the parasite from the gametocyte to sporozoite stage in the body of the mosquito is called extrinsic incubation period. The morphological cycle of maturation of gametocytes somewhat corresponds to the period of infectivity to mosquitoes. A single mosquito may remain infective throughout its life. Such a Institute of Life Long Learning, University of Delhi 22

24 synchronization of gametocyte and mosquito cycles have a marked selective advantage making sure that the parasite is transferred to the vertebrate host and the life cycle continues. Value addition: Video Heading text: Plasmodium Life cycle Body text: Life cycle of Plasmodium Source: Value addition: Activity (choose the correct option) Heading text: Let s check our understanding about the life cycle of malarial parasite. Body text: Source: self created (author) Pathogenecity All of the pathology of malaria is due to erythrocyte destruction by multiplying parasites. The disease process in malaria occurs due to: Local and systemic response of the host to parasitic antigens. Institute of Life Long Learning, University of Delhi 23

25 Tissue hypoxia caused by reduced oxygen delivery because of obstruction of blood flow by the parasitized erythrocytes. Anemia caused by destruction of large number of red cells. Incubation period: The symptoms do not appear immediately after humans get infected with Plasmodium sporozoites. The time interval between the infective bite of mosquito and onset of clinical symptoms is called the incubation period. During this period, sporozoites reach liver for exoerothrocytic schizogony followed by erythrocytic schizogony in RBC to produce sufficient erythrocytic merozoites to cause clinical symptoms. The incubation period in P.vivax varies from 8-31 days. Initial clinical symptoms: The initial signs and symptoms of include: headache, fever, shivering, joint pain, vomiting, hemolytic anemia, low blood pressure, jaundice, anorexia, hemoglobin in the urine, retinal damage and convulsions. The initial symptoms of malaria are flu-like and can resemble other conditions such as septicemia, gastroenteritis, and viral diseases and it is for this reason that malaria is frequently called The Great Imitator. Fig.16. Symptoms of Malaria Source: Main clinical manifestations: MALARIAL PAROXYSM Institute of Life Long Learning, University of Delhi 24

26 ANAEMIA HEPATOSPENOMEGALY Malarial paroxysm: It is the classical manifestation of acute malaria characterized by fever, chill, and rigor. Fever is the key clinical manifestation caused by the release of merozoites (asexual schizogonic developmental cycle) by rupture of schizont-infected red blood cell. Fever is irregular and does not show any distinct periodicity pattern. It lasts for 2 hrs to 6 hrs. High fever reaching 40.6 degree C (105 0 F), dry skin and often headache, nausea and vomiting are present in this stage. High Fever (HYPERPYREXIA) may also be characterized by incontinence of urine and feces mental disorientation, delirium and coma.the timing of fever cycle is 48 hours (tertian fever) in P. vivax and P. ovale infections. Each paroxysm shows a succession of three stages: 1.COLD STAGE: This is characterized by sudden onset of fever with rigor and sensation of extreme cold. Patient desires to be covered with blankets. This stage lasts for 15 minute and one hour. 2.HOT STAGE: Temperature may rise to 41 degree C (106 degree F). Patient feels burning hot and casts off his clothes. There is intense headache. This stage lasts from 2 to 6 hours. Chill subsides. 3.SWEATING STAGE: Fever comes down with profuse sweating. This stage lasts from 2 to 4 hours. Temperature drops rapidly to normal and skin is cool and moist. The classical 3 stages (cold, hot and sweating) may not always be observed due to maturation of generations of the parasite at different times. Value addition: Did you know? Heading text: Types of Malaria Body text: 1.TERTIAN, BENIGN TERTIAN OR VIVAX MALARIA: The causative agent of this type of Malaria is P. vivax. It is characterized by the recurrence of fever every third day, i.e., after 48 hours. This type of malaria does not result in death of the patient. The incidence of the disease is worldwide, mainly in temperate regions. 2.QUARTAN MALARIA: P. malariae is the causative organism of quartan malaria, which is characterized by the recurrence of fever every fourth day, i.e., at intervals of 72 hours. It is well known for its longevity, 40 years or more in untreated persons. Though it ordinarily does not prove fatal to the patient, the chronic infections sometimes give rise to lethal kidney conditions. The disease is of worldwide occurrence, but it is mainly confined to tropical and subtropical regions. 3.OVALE OR MILD TERTIAN MALARIA: This type of malaria resembles very much to the tertian malaria and is caused by P. ovale. The fever recurs every third day or at a intervals of 48 hours. Ovale malaria is not greatly harmful and is mainly confined to tropical Africa. 4.AESTIVO-AUTUMNAL, MALIGNANT TERTIAN OR PERNICIOUS MALARIA: is P. falciparum. In this also the fever cycle is of 48 hours. The fever is often fatal to Institute of Life Long Learning, University of Delhi 25

27 the patient as it affects the brain. Its incidence follows that of P. vivax. 5.QUOTIDIAN MALARIA: When more than one species of plasmodium infect the patient, or when 2 or 3 generations of parasites mature on successive days, the fever is repeated daily with an interval of 24 hours. Fig.Tertian and Quatran fever patterns in Malaria Source: Source: ILLL in house Anemia: It is the most immediate pathologic consequence of malaria.reasons for origin of anemia are: Destruction of erythrocytes on liberation of merozoites. Suppression of red cell production in the bone marrow. Infected erythrocytes become more fragile, rupture easily and are destroyed. The enlarged spleen due to malarial infection releases a lytic substance, lysolecithin, which destroys erythrocytes. Malarial parasites produce haemolysin (an antibody), which brings about haemolysis of some normal erythrocytes. Hepatosplenomegaly: Liver becomes enlarged as kupffer cells increases in number and their cytoplasm is filled with parasites, malarial pigment and cellular debris. Spleen is markedly enlarged (SPLENOMEGALY). In acute infection, spleen is soft with a thin capsule moderately enlarged and the splenic substance is congested. But in chronic disease, spleen is enlarged, hard with thick capsule. Red cell debris and leucocytic debris are found in spleen. There is an increase in the number of splenic macrophages (MACROPHAGES HYPERPLASIA). In chronic cases, spleen becomes enlarged and fibrosed so much that it ruptures under the influence of trauma. In chronic infections the spleen continues to enlarge (splenomegaly), Institute of Life Long Learning, University of Delhi 26

28 becoming hard and blackened in color due to the accumulation of malaria pigment, hemozoin. Relapse: In P. vivax and P.ovale malaria has the capacity to relapse, that is, there can be a reappearance of parasites in the blood after the patient has been treated and seemingly recovered. This type of relapse, called recurrence, is due to the delayed liberation of merozoites from exo-erythrocytic schizonts in the liver, called hypnozoites. It may also occur due to secondary exo-erythrocytic cycle. Relapse commonly occurs between 8 24 weeks and is commonly seen with P. vivax and P. ovale infections. It mainly occurs in temperate and tropical areas of Central and South America, southern Asia and the Indian subcontinent. It accounts for about 45% of all malaria cases. Parasites are often difficult to demonstrate in the peripheral blood film during relapses. Value addition: Did you know? Heading text: Recrudescence Body text: Recrudescence: Typically observed in P. falciparum and in P.malariae infection. It is the renewed appearance of observable symptoms in the host due to persistence of erythrocytic forms into the circulation. After a number of paroxysms, primary attack subsides with the development of partial immunity in the host. This is followed by a period of LATENCY during which there is no clinical illness or sometimes-even PARASITAEMIA. The parasites are not, however, eliminated at this stage, but persist in some erythrocytes, though the level of parasitaemia is below the fever threshold, or even below the microscopic threshold at times. Erythrocytic schizogony continues in the body at low levels and gradually the number of parasites increases in number to cross the fever threshold. Fresh malarial attacks then develop. These new malarial attacks that appear after a period of latency, usually within 8 weeks after the culmination of primary attack, and resulting from persistence of the erythrocytic cycle of the parasite are called RECRUDESCENCES. Recrudescence may be due to (1) Waning immunity of host, (2) Possibly to antigenic variations in the parasite evading themselves from the immune responses of host (3) An inadequate treatment with anti-malarial drugs. (4) Drug resistant persons There may be several such recrudescenes, which are milder than the primary attack. After varying number of such attacks, infection is eliminated in P. falciparum and P. malariae infections. Source: ILLL in house Epidemiology Institute of Life Long Learning, University of Delhi 27

29 Malaria is endemic throughout most of the tropics and subtropics of the world. Out of the approximately three billion people living in 106 countries who are exposed to malarial parasite, approximately 243 million will develop symptomatic malaria annually. P. vivax is most prevalent in 95 countries in tropical, sub-tropical and temperate regions,(guerra et al., 2009) except where there is a natural absence of anopheline mosquitoes (east of Vanuatu in the South Pacific) or among populations lacking the Duffy receptor on red cells (in much of Africa)(Baird 2008). It is only vivax malaria that occurs in the temperate latitudes up to the Korean peninsula and across the southern temperate latitudes of Asia to the Mediterranean Sea (Baird 2008). Approximately 2.6 billion people are at risk of infection with P. vivax malaria, and the ten countries with the highest estimated population at risk, are India>China>Indonesia>Pakistan>Vietnam>Philippines>Brazil>Myanmar>Thailand>Ethiopi a.( Guerra et al., 2009; Baird 2008) Estimates of annual infections range from 70 to 390 million, with about 80% occurring in South and Southeast Asia.( Baird 2008; Price et al., 2007) Approximately 10-20% of the world's cases of P. vivax infection occur in Africa, south of the Sahara.(Price et al., 2007) In eastern and southern Africa, 10% of malaria cases are due to P. vivax, whereas it accounts for <1% of cases in western and central Africa.( Mendis et al., 2001) Outside of Africa, P. vivax accounts for >50% of all malaria cases and about 80-90% of P. vivax outside of Africa occurs in the Middle East, Asia, and the Western Pacific and 10-15% in Central and South America.( Mendis et al., 2001). Value addition: Did you know? Heading text: Malaria Endemicity Body text: Holoendemic: transmission occurs all year long Hyperendemic: intense, but with periods of no transmission during dry season. Mesoendemic: regular seasonal transmission Hypoendemic: very intermittent transmission. Source: ocw.jhsph.edu/courses/malariology/pdfs/lecture3.pdf Mode of transmission Infective stage Sporozoites produced by sporogony in gut of female Anopheles mosquito. Merozoites produced by schizogony in humans. Source of infection Sporozoites are introduced into the vertebrate host during the blood meal of infected female Anopheles mosquito. Transmission ways The bite of a female Anopheles spp mosquito, which occurs mainly between dusk and dawn introduces the parasite in human leading to malaria. Congenital malaria caused by transmission of erythrocytic asexual forms of parasite to foetus via placenta (rare mechanism mainly found in endemic areas). Institute of Life Long Learning, University of Delhi 28

30 Transfusion malaria occurs by blood transfusion, sharing of contaminated needles, and organ transplant. Factors Affecting Transmission Distribution and abundance of the mosquito vector. Temperature and extent of water for larval breeding. Seasonal fluctuation of mosquito populations. Duration of conditions suitable for mosquito survival Susceptible population Susceptible populations are the ones with no or little immunity against the disease. In areas with high transmission (such as Africa south of the Sahara), the most vulnerable groups are: Young children, who are yet to develop immunity to malarial parasite. Pregnant women with decreased immunity. Travelers or migrants from areas free from malaria. In areas with lower transmission (such as Latin America and Asia), residents are less frequently infected hence with low immunity. Prophylaxis A. Treatment of infected individuals Individuals infected with malarial parasite should be treated with appropriate anti-malarial drugs (depending on the type of infection). B. Vector control 1. Destruction of adult mosquitoes. Adult mosquitoes can be most effectively combated in dwellings. The following methods can be employed to kill them: (a) KILLING BY HANDS. The mosquitoes, which approach or bite, may be killed by hands to save one as well as others. (b) TRAPS. These are small boxes made of wire gauze and internally lined by black paper or cloth. The mosquitoes tend to enter the boxes and then can be killed by closing them. (c) FUMIGATION. Mosquitoes can be driven out of the houses or killed by fumigation. sulphur, pyrethrum, cresol, tarcamphor or other derivaties of naphtha are burnt to produce poisonous fumes. Smoke of garlic is now considered to be effective in driving the mosquitoes out. (d) SPRAYING. Mosquitoes can be killed inside human dwellings by using mosquitobombs or by spraying DDT, flit, pyrethrum and other insecticides. (e) STERILIZATION. Sterilization of mosquitoes is now being achieved in some parts of the world, particularly Japan. This method is very effective in controlling mosquito population. 2. Source Reduction Institute of Life Long Learning, University of Delhi 29

31 It involves permanent destruction of mosquito breeding sites: Swampy areas, marshes and stagnant waters, such as pools, ponds, pits and ditches must be drained off as they are breeding places of mosquitoes. If possible, the breeding grounds may be filled up with earth or stones, etc. Eliminating receptacles that hold water, such as tin cans, buckets, cisterns, barrels, etc., can largely control domestic species. Bushes and shrubs should be cleared off. Open drains should be closed or made underground. Removing or covering standing water in vessels and rain barrels around houses. Removing or changing water from air coolers. 3. Chemical larviciding Oils may be applied to the water surface, suffocating the larvae and pupae. Oil solutions or emulsions of DDT, DDD and benzene hexachloride are effective larvicides. Dusts containing Paris green, DDT or BHC are effective in the control of surface feeding Anopheles larvae. Larger bodies of water can be sprayed with these poisonous substances by aeroplanes. Extracts of aquatic weed Ceratophyllum demensum control mosquito larvae in ponds. When tulsi (Oscimum sanctum) seeds are introduced in water they swell and the mucilage coating attracts and traps the mosquito larvae, which eventually die. Toxins from the bacterium Bacillus thuringiensis var. israelensis (Bti) can be applied in the same way as chemical insecticides. They are very specific, affecting only mosquitoes, black flies, and midges. Insect growth regulators such as methroprene are specific to mosquitoes and can be applied in the same way as chemical insecticides. 4. Biological control Larvicidal fishes, like stickle-backs, minnows (Gambusia) and trouts, etc., ducks and aquatic nymphs and adult insects like dragon-flies, which are natural enemies of mosquito, may be introduced in ornamental fountains, ditches, ponds, lakes, canals, tanks, etc. They feed upon mosquito larvae and pupae. Potential biological control agents, such as fungi (e.g., Laegenidium giganteum) or mermithid nematodes (e.g., Romanomermis culicivorax), parasitize and kill larval mosquitoes but they are not efficient for mosquito control and are not widely used. C. Prevention from mosquito bite Wearing protective light-colored long clothes that cover as much of the skin as possible. Use of mosquito net or Insecticide-treated bed nets while sleeping. Use of mosquito repellant. Use of anti-mosquito creams (repellents) on exposed parts of the bod Building houses on high grounds. Screening of doors, windows and ventilators. Treatment Treatment of vivax malaria Blood infection stage: Chloroquine remains the treatment of choice for vivax malaria. Institute of Life Long Learning, University of Delhi 30

32 Liver infection stage: At least a 14-day course of primaquine is required for the radical treatment (elimination of hypnozoites) of P.vivax. Eradication of the liver stages is achieved by giving primaquine, after checking the patients G6PD (lucose-6- phosphate dehydrogenase deficiency) status to reduce the risk of haemolysis. Chloroquine resistance vivax malaria: Amodiaquine, mefloquine and quinine are effective in the treatment of chloroquine-resistant P.vivax malaria. ACTs based on either amodiaquine, mefloquine or piperaquine, rather than monotherapy, are the recommended treatment of choice. These may also be used for P. vivax malaria in combination with primaquine for radical cure. Value addition: Did you know? Heading text: Classification of Antimalarial drugs Body text: The currently available antimalarials fall into 4 broad groups: 1. Quinoline-related: quinine, chloroquine, amodiaquine, halofantrine, mefloquine and primaquine, as well as the newer aryl amino-alcohol, lumefantrine 2. Anti-fols : pyrimethamine proguanil or Lapudrine, chlorproguanil, trimethoprim and dapsone 3. Ubiquinone analogs: atovaquone 4. Artemisinin compounds: artemisinin, artemether, arteether and artesunate Anti-malarial drugs can be classified on the basis of type of action: 1. Suppressive - effective on asexual blood stages and limits clinical symptoms. 2. Therapeutic - effective on asexual blood stages in the acute stage. 3. Radical cure - effective on extra-erythrocytic forms. 4. Gametocidal - effective on the gametes. 5. Sporonticidal - effective on the sporozoites in the mosquito and makes them noninfective to the vertebrate host. Source: Value addition: Did you know? Heading text: The oldest yet least understood antimalarial drug! Body text: Primaquine is one of the oldest of currently used anti-malarial drugs an is the least well understood. This drug was first deployed to prevent P. vivax relapses i US soldiers returning to the United States from World War II. Primaquine destroys th liver forms of all plasmodium species but toxicity considerations preclude its widesprea use in the control of malaria. Primaquine can also precipitate acute and dangerou intravascular haemolysis in subjects with glucose-6-phosphate dehydrogenase (G6PD deficiencies, who are most frequently found in malaria-endemic countries. Source: Institute of Life Long Learning, University of Delhi 31

33 Summary There are three distinct asexual replicative stages of malaria parasites - exoerythrocytic schizogony, blood stage schizogony, and sporogony, which results in the production of invasive, forms -merozoites and sporozoites. All invasive stages have apical organelles, which is typical characteristic of apicomplexan species. Different invasive stages invade different types of cells or tissues and their motility also varies. After the successful invasion of host, a few of these invasive forms show high multiplication to establish the infection in the new host. During the blood meal, sporozoites are injected with the saliva of a feeding mosquito into the host thus initiating the infection. The circulatory system of the host carries the sporozoites to the liver and where they invade hepatocytes. Within the hepatocyte, the intracellular parasite replicates asexually called exoerythrocytic schizogony. Exoerythrocytic schizogony results in the production of merozoites, which are released into the bloodstream. In case of P. vivax, a proportion of the liver-stage parasites remain in a dormant period inside the liver cells instead of immediately undergoing asexual replication. They are called hypnozoites. Hypnozoites, which are responsible for, relapses, reactivates after several weeks to months (or years) after the primary infection. Merozoites invade erythrocytes and undergo a trophic period in which the parasite enlarges. Due to ring like appearance, the early trophozoite is often referred to as 'ring form'. Trophozoite enlargement occurs by an active metabolism inside host cell, which includes the ingestion of host cytoplasm and the proteolysis of hemoglobin into amino acids. A schizont is formed at the end of the trophic period by multiple rounds of nuclear division without cytokinesis. Merozoites also called a segmenter bud out of the mature schizont, which are then released following rupture of the infected erythrocyte. Institute of Life Long Learning, University of Delhi 32

34 These merozoites now enter fresh erythrocytes thus reinitiates another round of the bloodstage replicative cycle. Periodic onset of fevers in malaria is due to the synchronous rupture of infected erythrocytes and release of merozoites. The parasite can also differentiate into sexual forms known as macro- or microgametocytes instead of undergoing asexual replicative cycle. A sexual reproduction occurs as the parasite switches from vertebrate to invertebrate host resulting in invasive ookinete. Ingestion of gametocytes by the mosquito vector sets in gametogenesis (i.e., the production of gametes) thus they escape from the host erythrocyte. A drop in temperature, an increase in carbon dioxide, and mosquito metabolites may induce gametogenesis. Microgametes are flagellated forms, formed by the process of exflagellation, which fertilizes the macrogamete leading to the formation of a zygote. The zygote develops into a motile ookinete, which penetrates the gut epithelial cells of mosquito and develops into an oocyst. Multiple rounds of asexual replication of the oocyst results in the production of sporozoites. Sporozoites are released into the hemocoel (body cavity) of the mosquito by rupturing of the mature oocyst. The sporozoites then migrate to and invade the salivary glands of the mosquito, thus completing the life cycle. Summary of prevention and control: Institute of Life Long Learning, University of Delhi 33

35 Fig. 17.Summarized steps for control of Malaria Source: ILLL in house Institute of Life Long Learning, University of Delhi 34